RESUMO
INTRODUCTION: We previously reported that TRPM7 regulates glioma cells' stemness through STAT3. In addition, we demonstrated that FOSL1 is a response gene for TRPM7, and the FOSL1 gene serves as an oncogene to promote glioma proliferation and invasion. METHODS: In the present study, we determined the effects of FOSL1 on glioma stem cell (GSC) markers CD133 and ALDH1 by flow cytometry, and the maintenance of stem cell activity by extreme limiting dilution assays (ELDA). To further gain insight into the mechanism by which TRPM7 activates transcription of the FOSL1 gene to contribute to glioma stemness, we constructed a FOSL1 promoter and its GAS mutants followed by luciferase reporter assays and ChIP-qPCR in a glioma cell line and glioma patient-derived xenoline. We further examined GSC markers ALDH1 and TRPM7 as well as FOSL1 by immunohistochemistry staining (IHC) in brain tissue microarray (TMA) of glioma patients. RESULTS: We revealed that FOSL1 knockdown reduces the expression of GSC markers CD133 and ALDH1, and FOSL1 is required to maintain stem cell activity in glioma cells. The experiments also showed that mutations of - 328 to - 336 and - 378 to - 386 GAS elements markedly reduced FOSL1 promoter activity. Constitutively active STAT3 increased while dominant-negative STAT3 decreased FOSL1 promoter activity. Furthermore, overexpression of TRPM7 enhanced while silencing of TRPM7 reduced FOSL1 promoter activity. ChIP-qPCR assays revealed that STAT3, present in nuclear lysates of glioma cells stimulated by constitutively activated STAT3, can bind to two GAS elements, respectively. We demonstrated that deacetylation of FOSL1 at the Lys-116 residue located within its DNA binding domain led to an increase in FOSL1 transcriptional activity. We found that the expression of TRPM7, ALDH1, and FOSL1 protein is associated with grades of malignant glioma, and TRPM7 protein expression correlates to the expression of ALDH1 and FOSL1 in glioma patients. CONCLUSIONS: These combined results demonstrated that TRPM7 induced FOSL1 transcriptional activation, which is mediated by the action of STAT3, a mechanism shown to be important in glioma stemness. These results indicated that FOSL1, similar to GSC markers ALDH1 and TRPM7, is a diagnostic marker and potential drug target for glioma patients.
Assuntos
Glioma , Canais de Cátion TRPM , Humanos , Canais de Cátion TRPM/genética , Oncogenes , Bioensaio , Encéfalo , Glioma/genética , Proteínas Serina-Treonina Quinases , Fator de Transcrição STAT3/genéticaRESUMO
BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.
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Transplante de Células/métodos , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/isolamento & purificação , Potenciais de Ação/fisiologia , Animais , Biomarcadores , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citometria de Fluxo/métodos , Humanos , Camundongos , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Nanotecnologia , Conformação de Ácido Nucleico , Células-Tronco Pluripotentes/fisiologia , Sondas RNA/química , Sondas RNA/isolamento & purificação , RNA Mensageiro/química , Troponina I/genética , Troponina T/genéticaRESUMO
OBJECTIVE: To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. METHODS AND RESULTS: mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D-null, ionomycin-treated, and reactive oxygen species-treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca(2+)(mit)) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca(2+)(mit) increased significantly in strongly stimulated platelets. Abrogation of Ca(2+)(mit) entry, either by inhibition of the Ca(2+)(mit) uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. CONCLUSIONS: Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca(2+)(mit) levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca(2+)(mit) entry allows the specific inhibition of platelet procoagulant activity.
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Plaquetas/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Humanos , Técnicas In Vitro , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Modelos Animais , Transdução de Sinais/fisiologia , Trombina/farmacologiaRESUMO
Each year, tens of thousands of children undergo cardiopulmonary bypass (CPB) to correct congenital heart defects. Although necessary for surgery, CPB involves stopping the heart and exposing it to ischemic conditions. On reoxygenation, the heart can experience effects similar to that of acute myocardial infarction. Although much is known about adult injury, little is known about the effects of global ischemia on newborn ventricles. We studied newborn (2 to 4 days old) and adult (>8 weeks old) rabbit hearts subjected to ischemia-reperfusion (30 min of ischemia and 60 min of reperfusion). Our data demonstrated chamber- and age-specific changes in oxidative stress. During ischemia, hydrogen peroxide (H(2)O(2)) increased in both right-ventricular (RV) and left-ventricular (LV) myocytes of the newborn, although only the RV change was significant. In contrast, there was no significant increase in H(2)O(2) in either RV or LV myocytes of adults. There was a fivefold increase in H(2)O(2) formation in newborn RV myocytes compared with adults (P = 0.006). In whole-heart tissue, superoxide dismutase activity increased from sham versus ischemia in the left ventricle of both adult and newborn hearts, but it was increased only in the right ventricle of the newborn heart. Catalase activity was significantly increased after ischemia in both adult ventricles, whereas no increase was seen in newborn compared with sham hearts. In addition, catalase levels in newborns were significantly lower, indicating less scavenging potential. Nanoparticle-encapsulated ebselen, given as an intracardiac injection into the right or left ventricle of newborn hearts, significantly increased functional recovery of developed pressure only in the right ventricle, indicating the potential for localized antioxidant therapy during and after pediatric surgical procedures.
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Ventrículos do Coração/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antioxidantes/administração & dosagem , Azóis/administração & dosagem , Catalase/biossíntese , Ventrículos do Coração/metabolismo , Peróxido de Hidrogênio/metabolismo , Isoindóis , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica/metabolismo , Nanopartículas , Compostos Organosselênicos/administração & dosagem , Coelhos , Recuperação de Função FisiológicaRESUMO
Resilience, the ability to overcome stressful conditions, is found in most mammals and varies significantly among individuals. A lack of resilience can lead to the development of neuropsychiatric and sleep disorders, often within the same individual. Despite extensive research into the brain mechanisms causing maladaptive behavioral-responses to stress, it is not clear why some individuals exhibit resilience. To examine if sleep has a determinative role in maladaptive behavioral-response to social stress, we investigated individual variations in resilience using a social-defeat model for male mice. Our results reveal a direct, causal relationship between sleep amount and resilience-demonstrating that sleep increases after social-defeat stress only occur in resilient mice. Further, we found that within the prefrontal cortex, a regulator of maladaptive responses to stress, pre-existing differences in sleep regulation predict resilience. Overall, these results demonstrate that increased NREM sleep, mediated cortically, is an active response to social-defeat stress that plays a determinative role in promoting resilience. They also show that differences in resilience are strongly correlated with inter-individual variability in sleep regulation.
To many of us, it may seem obvious that sleep is restorative: we feel better after a good night's rest. However, exactly how sleep benefits the brain and body remains poorly understood. One clue may lie in neuropsychiatric disorders: these conditions such as depression and anxiety are often accompanied by disrupted sleep. Additionally, these neuropsychiatric disorders are frequently caused or worsened by stress, which can also interfere with sleep. This close association between stress and sleep has led some to hypothesize that sleep serves to overcome the adverse effects of stress on the brain, but this hypothesis remains largely untested. One type of stress that is common to all mammals is social stress, defined as stress caused by social interactions. This means that mice and other rodents can be subjected to social stress in the laboratory to test hypotheses about the effects of stress on the brain. Importantly, in both animals and humans, there are individual differences in resilience, or the ability to overcome the adverse effects of stress. Based on this information, Bush et al. set out to establish whether sleep can regulate resilience to social stress in mice. When the mice were gently kept awake during their normal sleep time, resilience decreased and so the mice were less able to overcome the negative effects of stress. Conversely, increasing sleep, by activating an area of the brain responsible for initiating sleep, increased the mice's resilience to social stress. Thus, Bush et al. showed that changes in sleep do lead to changes in resilience. To find out whether resilience can be predicted by changes in sleeping patterns, Bush et al. studied how both resilient mice and those susceptible to stress slept before and after social stress. Resilient mice would often sleep more after social stress; meanwhile, few changes were observed in susceptible mice. Surprisingly, sleep quality also predicted resilience, with resilient mice sleeping better than susceptible mice even before exposure to social stress. To determine whether the differences in sleep that predict resilience can be detected as brain activity, Bush et al. placed electrodes in two regions of the prefrontal cortex a part of the brain important for decision-making and social behaviors to measure how mice recovered lost sleep. This experiment revealed that the changes in sleep that predict resilience are prominent in the prefrontal cortex. Overall, Bush et al. reveal that sleeping more and sleeping better promote resilience to social stress. Furthermore, the results suggests that lack of sleep may lead to increased risk of stress-related psychiatric conditions. Humans are one of the few species that choose to deprive themselves of sleep: Bush, et al. provide evidence that this choice may have significant consequences on mental health. Furthermore, this work creates a new model that lays the groundwork for future studies investigating how sleep can overcome stress on the brain.
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Movimentos Oculares , Estresse Psicológico , Animais , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Estresse Psicológico/psicologia , Córtex Pré-Frontal , Sono , MamíferosRESUMO
We have reported that transient receptor potential melastatin-related 7 (TRPM7) regulates glioma stem cells (GSC) growth and proliferation through Notch, STAT3-ALDH1, and CD133 signaling pathways. In this study, we determined the major contributor(s) to TRPM7 mediated glioma stemness by further deciphering each individual Notch signaling. We first determined whether TRPM7 is an oncotarget in glioblastoma multiforme (GBM) using the Oncomine database. Next, we determined whether TRPM7 silencing by siRNA TRPM7 (siTRPM7) induces cell growth arrest or apoptosis to reduce glioma cell proliferation using cell cycle analysis and annexin V staining assay. We then examined the correlations between the expression of TRPM7 and Notch signaling activity as well as the expression of GSC markers CD133 and ALDH1 in GBM by downregulating TRPM7 through siTRPM7 or upregulating TRPM7 through overexpression of human TRPM7 (M7-wt). To distinguish the different function of channel and kinase domain of TRPM7, we further determined how the α-kinase-dead mutants of TRPM7 (α-kinase domain deleted/M7-DK and K1648R point mutation/M7-KR) affect Notch activities and CD133 and ALDH1 expression. Lastly, we determined the changes in TRPM7-mediated regulation of glioma cell growth/proliferation, cell cycle, and apoptosis by targeting Notch1. The Oncomine data revealed a significant increase in TRPM7 mRNA expression in anaplastic astrocytoma, diffuse astrocytoma, and GBM patients compared to that in normal brain tissues. TRPM7 silencing reduced glioma cell growth by inhibiting cell entry into S and G2/M phases and promoting cell apoptosis. TRPM7 expression in GBM cells was found to be positively correlated with Notch1 signaling activity and CD133 and ALDH1 expression; briefly, downregulation of TRPM7 by siTRPM7 decreased Notch1 signaling whereas upregulation of TRPM7 increased Notch1 signaling. Interestingly, kinase-inactive mutants (M7-DK and M7-KR) resulted in reduced activation of Notch1 signaling and decreased expression of CD133 and ALDH1 compared to that of wtTRPM7. Finally, targeting Notch1 effectively suppressed TRPM7-induced growth and proliferation of glioma cells through cell G1/S arrest and apoptotic induction. TRPM7 is responsible for sustained Notch1 signaling activation, enhanced expression of GSC markers CD133 and ALDH1, and regulation of glioma stemness, which contributes to malignant glioma cell growth and invasion.
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The circadian clock is an evolutionarily, highly conserved feature of most organisms. This internal timing mechanism coordinates biochemical, physiological and behavioral processes to maintain synchrony with the environmental cycles of light, temperature and nutrients. Several studies have shown that light is the most potent cue used by most organisms (humans included) to synchronize daily activities. In mammals, light perception occurs only in the retina; three different types of photoreceptors are present within this tissue: cones, rods and the newly discovered intrinsically photosensitive retinal ganglion cells (ipRGCs). Researchers believe that the classical photoreceptors (e.g., the rods and the cones) are responsible for the image-forming vision, whereas the ipRGCs play a key role in the non-image forming vision. This non-image-forming photoreceptive system communicates not only with the master circadian pacemaker located in the suprachiasmatic nuclei of the hypothalamus, but also with many other brain areas that are known to be involved in the regulation of several functions; thus, this non-image forming system may also affect several aspects of mammalian health independently from the circadian system.
Assuntos
Ritmo Circadiano/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Animais , Humanos , Modelos Biológicos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Opsinas de Bastonetes/fisiologiaRESUMO
AIMS: To evaluate the ability of N-acetylglucosamine (GlcNAc) decorated nanoparticles and their cargo to modulate calcium handling in failing cardiac myocytes (CMs). MATERIALS & METHODS: Primary CMs isolated from normal and failing hearts were treated with GlcNAc nanoparticles in order to assess the ability of the nanoparticles and their cargo to correct dysfunctional calcium handling in failing myocytes. RESULTS & CONCLUSION: GlcNAc particles reduced aberrant calcium release in failing CMs and restored sarcomere function. Additionally, encapsulation of a small calcium-modulating protein, S100A1, in GlcNAc nanoparticles also showed improved calcium regulation. Thus, the development of our bioactive nanoparticle allows for a 'two-hit' treatment, by which the cargo and also the nanoparticle itself can modulate intracellular protein activity.
Assuntos
Acetilglucosamina/administração & dosagem , Insuficiência Cardíaca/tratamento farmacológico , Miócitos Cardíacos/metabolismo , Nanopartículas/administração & dosagem , Acetilglucosamina/química , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Nanopartículas/química , Proteínas S100/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologiaRESUMO
Regulation of catalase (CAT) by peroxisome proliferator-activated receptor-γ (PPARγ) was investigated to determine if PPARγ activation provides cardioprotection from oxidative stress caused by hydrogen peroxide (H(2)O(2)) in an age-dependent manner. Left ventricular developed pressure (LVDP) was measured in Langendorff perfused newborn or adult rabbit hearts, exposed to 200µM H(2)O(2), with perfusion of rosiglitazone (RGZ) or pioglitazone (PGZ), PPARγ agonists. We found: (1) H(2)O(2) significantly decreased sarcomere shortening in newborn ventricular cells but not in adult cells. Lactate dehydrogenase (LDH) release occurred earlier in newborn than in adult heart, which may be due, in part, to the lower expression of CAT in newborn heart. (2) RGZ increased CAT mRNA and protein as well as activity in newborn but not in adult heart. GW9662 (PPARγ blocker) eliminated the increased CAT mRNA by RGZ. (3) In newborn heart, RGZ and PGZ treatment inhibited release of LDH in response to H(2)O(2) compared to H(2)O(2) alone. GW9662 decreased this inhibition. (4) LVDP was significantly higher in both RGZ+H(2)O(2) and PGZ+H(2)O(2) groups than in the H(2)O(2) group. Block of PPARγ abolished this effect. In contrast, there was no effect of RGZ in adult. (5) The cardioprotective effects of RGZ were abolished by inhibition of CAT. In conclusion, PPARγ activation is cardioprotective to H(2)O(2)-induced stress in the newborn heart by upregulation of catalase. These data suggest that PPARγ activation may be an effective therapy for the young cardiac patient.